A protein kinase plays a key role in mediation of signal transduction via phosphorylation of a hydroxyl group present in a tyrosine, serine or threonine residue of a protein, and, thus, is deeply involved in the regulation of cell growth, differentiation, proliferation, etc.
As is well known, a balance between the “on-state” and the “off-state” of an intracellular signaling pathway is essential for maintenance of homeostasis of a cell. When a normal intracellular signaling pathway is interrupted due to overexpression or mutation of a specific protein kinase (e.g., mostly continuation of the “on-state” of intracellular signals), it may cause various diseases such as cancer, inflammatory diseases, metabolic diseases and brain diseases. It is estimated that human genome contains 518 protein kinases which constitute approximately 1.7% of all human genes [Manning et al., Science, 298, (2002), 1912]; and the protein kinases can be divided into two major types: tyrosine protein kinases (90 species or more) and serine/threonine protein kinases. The tyrosine protein kinases can be divided into 58 species of receptor tyrosine kinases which can be further categorized into 20 subtypes, and 32 species of cytoplasmic/non-receptor tyrosine kinases which can be further categorized into 10 subtypes. A receptor tyrosine kinase has a kinase domain that can receive a growth factor on the surface of a cell, and an active site for phosphorylation of a tyrosine residue in cytoplasm. When a growth factor binds to the extracellular domain of the growth receptor, the receptor tyrosine kinase forms a polymer, which may result in autophosphorylation of specific tyrosine residues in the cytoplasmic active site. This may trigger a cascade of phosphorylation of downstream intracellular protein kinases that ultimately transmits the extracellular signal into the nucleus, thereby causing transcription of various genes and synthesis of various proteins that may be involved in cell growth, differentiation, proliferation and the like. In this process, it is known that if protein kinases are abnormally overexpressed or abnormally activated by mutation, this may induce various kinds of diseases such as cancer.
Among the various cytoplasmic kinases, Raf is one of the kinases that participate in mitogen-activated protein kinase (MAPK) pathway where a signal is initiated by the receptor protein kinase activated by a growth factor and transmitted via Ras-Raf-MEK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) [Solit, D. B. et al., Nature, 439, (2006), 358]. Currently, it is known that Raf has three types of isoforms, i.e., A-Raf, B-Raf and C-Raf [Jansen H W, et al., EMBO. J., 2, (1983), 1969; Marais R. et al., Cancer Surv., 27, (1996), 101]. Since abnormal activation in the MAPK pathway has been observed in approximately 30% of human cancer tissues and gene mutation of B-Raf and C-Raf showing aberrant activation has been confirmed in cancer tissues, it is generally accepted that Raf plays a very important role in the MAPK pathway of cancer tissues.
Accordingly, there have been suggested methods of treating cancer by using a compound having an inhibitory effect against abnormal activities of Raf kinases. Hence, several Raf and modified Raf kinase inhibitors are being clinically used or currently under development for the treatment of cancer. Examples of such Raf kinase inhibitors include: sorafenib (Nexava®, Bayer) which is used for treatment of liver cancer, vemurafenib (PLX-4032, RG7204, Roche) which has been recently approved for treatment of melanoma, and drugs that are currently being tested in clinical trials such as regorafenib and RDEA119 by Bayer, RAF265 by Novartis, E3810 by Advan Chem, DCC2036 by Deciphera Pharma., CKI-27 by Chugai Pharma., RO-5126766 by Roche, and the like.
However, it is observed that many cancers develop a tolerance to said inhibitors. It has been postulated that the tolerance is caused by abnormal activation of MAPK pathway due to mutation of Raf, activation of complementary signaling system among different Raf isoforms, different signal transduction pathway depending on the subtypes (i.e., K-Ras, N-Ras and H-Ras) of Ras, which is a upstream protein of MAPK signal transduction, and, moreover, activation of signaling systems other than MAPK due to mutation of Ras.
In this connection, it has been proven that inhibition of Raf/MEK/ERK signaling system to suppress cell proliferation and inhibition of VEGFR2/PDGFR-β to suppress tumor angiogenesis may be effective against diseases caused by mutations in Raf and Ras [Allen, E. et al., Clin. Cancer Res., 17, (2011), 5299].